The present invention relates to the field of biophysical analysis of molecules. In particular, the present invention is useful for the identification and analysis of a molecule, such as a member of a combinatorial library, wherein the identified molecule has a demonstrated pharmacological or physiological activity.
Over the past ten years, there has been a growing demand for the production and identification of molecules that have pharmacological or other physiological activity as, for example, agonists or antagonists of various cellular acceptor molecules, such as cell-surface receptors, enzymes, or antibodies. Such molecules can be peptides, oligonucleotides, or other organic compounds, such as heterocyclics and the like, which are commonly the products of combinatorial synthesis, thus forming the members of combinatorial libraries. The unifying feature of these molecules is operational in that they bind specifically to known acceptors. In consequence of such binding, a physiological response occurs whereby certain biological processes are modulated, which can have applications in medicine and agriculture.
Searching for molecules that are useful in medical or veterinary applications, or in agriculture or agrobiology, entails (1) generating collections of such molecules, (2) screening such molecules for physiological activity, and (3) identifying the structure of molecules that provide a positive result in the screen. The first two steps can be accomplished using methods well-known in the art, some of which were discussed in Benkovic et al., PCT/US95/03355, which is incorporated herein in toto by reference. The subject matter of Benkovic et al. related to a mass spectrometric method for identification of such molecules, including those that are members of a combinatorial library. Covalent attachment of such molecules to a substrate, such as a polystyrene or other resins particle, via a suitable linker, thus forming a molecule-substrate complex, is commonly used for purposes of manipulating the molecules; however, for mass spectrometric identification of the molecules, the molecules must be separated from the substrate. One approach that has been used requires use of a photo- or acid-labile linker, however such an approach requires identification of linkers that include either a photo-labile or an acid-labile linkage and subjecting the substrate-attached molecules to a suitable wavelength of light or acid. Such a step requires extra manipulation of the samples, and consumes time. Secondly, the process used to cleave the covalent linkage between the molecule and the molecule-substrate complex may destroy or damage the substrate or the entire molecule-substrate complex, thus retesting of a given molecule requires having additional molecule-substrate complexes.
Accordingly, the analysis of a molecule covalently linked to a substrate, such as the molecules of a combinatorial library, or any collection of molecules so linked to a substrate or substrates, is necessarily impeded by the rate at which substrates, such as beads, having individual molecules attached thereto can be analyzed for the identity of the attached molecule. In view of the literally millions of candidate molecules to be screened in a given library, for example, it is probable that at least hundreds, if not thousands, of the molecule-attached beads would generate positive signals (including false positive signals) requiring further analysis. The limitation of being able to sequence only a few molecules per day, as has been reported by Lam et al., Nature, 354, 82 (1991), for example, therefore, presents a strong drawback to current strategies of screening collections of molecules, such as combinatorial libraries, for pharmaceutical compounds. Moreover, if a method allowed identification of a molecule included on a molecule-substrate complex with respect to molecular weight, more preferably with respect to structure, where the molecule was indicated in a screen as having a desireable characteristic, without having to remove such a molecule-substrate complex from the group of other such complexes, in the presence of which the molecule-substrate complex was screened, the procedure of screening and identifying molecules of interest would be greatly improved. Further yet, if a method required essentially a unified step of testing a molecule-substrate complex directly without causing damage to the complex but for the removal of a portion of the included molecule, the process would not only be faster, but would preserve the molecule-substrate complex having a then reduced amount of the molecule, which could be used for further analysis.
It has now been discovered that a mass spectrometric assay can be used to analyze molecules covalently attached to a substrate, such molecules being peptides, oligonucleotides, heterocyclic molecules, or other chemical species. Such molecules can be members of any collection of molecules including those isolated from natural sources or synthesized, such as those of a combinatorial library. Individual members of a collection of molecules, for example, can be constructed on or attached to a suitable substrate or substrates and screened, and the individual substrate or portion thereof that is identified as having a molecule that, for example, specifically interacts with an acceptor molecule of interest (i.e., positive screen result) can be identified in the presence of identical substrates having other unselected molecules attached thereto and subjected to mass spectrometric assay without removal from the total collection to determine the precise molecular weight of the selected molecule. A preferred aspect of the method includes the use of linking moieties or substrates having reactive groups attached thereto that covalently link the individual molecules of the collection to the substrate, whereby the linkage of at least a portion of the molecules linked to the substrate is cleaved without disturbing the molecule""s structure or the integrity of the substrate, allowing analysis of the free molecules and subsequent analysis of the remaining linked molecules. Consequently, the present invention greatly improves the ability of artisans of the relevant art to identify, for example, pharmaceutically active agents derived from collections of molecules, such as combinatorial libraries.
In particular, the present invention relates to a method of identifying a molecule of a molecule-substrate complex, wherein the molecule is covalently attached directly to a substrate or indirectly by means of a linking moiety, comprising:
(a) bombarding the molecule-substrate complex with energized particles to cleave the molecule from the molecule-substrate complex; and
(b) determining the molecular weight of the cleaved molecule by means of mass spectrometry. Preferably, the method further comprises irradiating the cleaved molecule with photons.
The present method is further directed to a molecule that is selected from the group consisting of amino acids, peptides, oligonucleotides, heterocyclic compounds, and combinations thereof. The substrate used in the context of the present invention preferably comprises a polymeric resin or a metal; and, in another embodiment, further comprises a linking moiety attached thereto. Preferably, the polymeric resin is a polystyrene resin having a linking moiety attached thereto.
The linking moiety used in the context of the present invention preferably comprises at least one reactive group that is selected from the group consisting of hydroxyl, amino, carboxyl, acetal, thioacetal, C1-C10 alkylamino, C1-C10 aralkylamino, and C1-C10 haloalkyl, and an o-nitrobenzylic group having a benzylic hydrogen. Preferably, the linking moiety is selected from the group consisting of F-moc-2,4-dimethoxy-4xe2x80x2-(carboxymethyloxy)-benzhydrylamine, F-moc-methoxy-4xe2x80x2(gamma-carboxypropyloxy)benzhydrylamine, p-alkoxybenzyl alcohol, benzylacetal, benzylthioacetal, benzhydrylamine, Clxe2x80x94CH2-Ph, 2-methoxy-4-alkoxy benzyl alcohol, and o-nitrobenzyloxy carbonyl. More preferably, the linking moiety is selected from the group consisting of 2-methoxy-4-alkoxy benzyl alcohol, benzylacetal, and benzylthioacetal.
The present method includes cleaving the molecule from the substrate in the molecule-substrate complex without substantial modification of the molecule or destruction of the substrate. Such cleaving is preferably accomplished by bombardment of the molecule-substrate complex with energized particles, wherein the particles are preferably gallium or argon. The particles are energized by subjection to an electric field of between about one and about 30 kilovolts. It is believed that the free molecule that results from the bombardment is charged or uncharged; the molecule becomes charged or further charged by irradiation by a laser beam.
The substrate used in the context of the present method is preferably a bead. A bead used in this context has a diameter of from about 10 microns to about 120 microns.
The mass spectrometry used in the present invention is preferably time-of-flight secondary ion mass spectrometry. The method further comprises mapping of the spatial distribution of the molecules on the aforementioned beads that, for example, are arranged on a grid.
In a preferred embodiment, the molecule subjected to analysis under the present invention is an amino acid or a peptide. Preferably, the peptide comprises two to ten amino acids. The method further comprises determination of the sequence of the peptide from the fragmentation pattern obtained in the mass spectrometry. Alternatively, the molecule subjected to analysis under the present invention is a heterocyclic compound comprising four to seven membered rings having N, S, or O, and combinations thereof.
A preferred embodiment of the present invention relates to a method of identifying a molecule of a molecule-substrate complex, wherein the molecule is covalently attached directly to a substrate or indirectly by means of a linking moiety, comprising:
(a) bombarding the molecule-substrate complex with energized particles to cleave the molecule from the molecule-substrate complex;
(b) irradiating the cleaved molecule with photons; and
(c) determining the molecular weight of the irradiated molecule by means of mass spectrometry, wherein the substrate is a polystyrene bead having a reactive group, the molecule is an amino acid, peptide, oligonucleotide, or a heterocyclic compound, or a combination thereof, the covalent bond is sensitive to energized particle bombardment, the energized particles are gallium atoms, the photon source is a laser, and the mass spectrometry is time-of-flight secondary ion mass spectrometry.
These and other features and advantages of the invention will be more readily apparent upon reading the following detailed description of the invention and upon reference to the accompanying drawings, all of which are given by way of illustration only, and are not limitative of the present invention.